Translocation of the HIV-1 genome into the nucleus of a target cell is a critical step in viral replication in both dividing and non-dividing cells. This process can be attributed to the interaction between the viral preintegration complex and the cellular nuclear import machinery. One of the main regulators of this interaction is viral protein R (Vpr). However, molecular details of Vpr's activity are not understood. In addition, nuclear import of HIV in some cell types appears to be Vpr-independent. Our recent studies have identified heat shock protein 70 (Hsp70) as a substitute for Vpr in such cells, but the molecular reason for this substitution is not known. Our long-range goal is to elucidate the mechanisms that govern the nuclear import of HIV-I. The objective of this application is to define the molecular mechanisms responsible for Vpr's activity in HIV-1 nuclear importation, and how Hsp70 substitutes for Vpr as the nuclear importer in some cell types. The central hypothesis of the application, formulated on the basis of strong preliminary data, is that Vpr (or Hsp70) critically regulates HIV-1 nuclear import by binding to karyopherin, the cellular receptor for proteins destined for the nucleus, thereby enhancing the affinity of karyopherin for the viral pre-integration complex. The rationale for the proposed research is that, once the mechanisms of Vpr/HspT0 activity are known, agents can be designed to inhibit HIV-1 nuclear import and thus abort infection at a pre-integration step. The central hypothesis will be tested and the objective of the application accomplished by pursuing three specific aims: 1) Identify cellular proteins critical for Vpr's activity during HIV-1 nuclear import; 2) Characterize the mechanism(s) of Vpr's nuclear import activity; and 3) Investigate the mechanism of activity of Hsp70 in (HIV-1) nuclear import and its relation to Vpr. The proposed work is innovative, as it capitalizes on our recent original findings of the Vpr/karyopherin interaction and its role in HIV-1 nuclear import, and will furnish important new information on the previously unknown activity of Hsp70 in nuclear import. It is our expectation that the proposed approach will reveal missing details on the interactions between Vpr and cellular proteins during HIV-1 nuclear import. These results will be significant, because they are expected to facilitate discovery of agents that would inhibit Vpr activity and thus abort HIV replication. Additionally, these studies will provide fundamental advances in the field of cellular biology.